CN115297880A - Topical composition comprising extract of crude drug comprising arillus longan for treating or improving skin ulcer, and its application - Google Patents

Abstract

The present invention relates to a topical pharmaceutical composition and a cosmetic composition for treating and improving skin ulcer, which comprise a combined crude drug extract of arillus longan, ligusticum, and polygala tenuifolia as an active ingredient.

Description

Topical composition comprising extract of crude drug comprising arillus longan for treating or improving skin ulcer, and its application

Technical Field

The present invention relates to a topical composition comprising a combined crude drug extract containing Arillus longan (longanaceae arilus) for treating or improving skin ulcer and its use.

Background

Skin wounds are induced by trauma, traumatic injury, pinning, etc., and Wound Healing is a process of restoring damaged tissue for the integrity of the Skin (Pazyar N, yaghoobi R, rafiee E, mehraban A, feily A (2014) Skin Wound Healing and photomedicine: A review. Skin Pharmacol physical. 27: pp.303-310.).

Wound Healing is achieved by a four-stage sequential continuous process of hemostasis, inflammation, proliferation and reformation (Demidova-Rice TN, hamblin MR, herman IM (2012) Acute and Impaired Wound Healing: pathophysiology and Current Methods for Drug Delivery, part 1 Normal and Chronic Wends.

The process is achieved by effective interaction among various cells, proteins, cytokines, is very complex and delicate, and if the wound healing process cannot be normally performed for various reasons, a chronic wound is formed.

For example, chronic wounds are known to cause abnormal increases In pro-inflammatory cytokines and Matrix Metalloproteinases (MMPs) if stasis induces an excessive inflammatory response during the inflammatory phase (wearing SA, krieg T, davidson JM (2007) In-migration In Wound Repair: molecular and Cellular mechanisms. Journal of Investigative surgery.127: 514-525.).

Furthermore, it also reduces the Expression of Growth Factors, failing to proceed normally with the process of vascularization and the formation of granulation tissue (Frank S, hubner G, breier G, longaker MT, greenhalgh DG, werner S (1995) Regulation of Vascular Endothelial Growth Factor Expression in filtered Ker-formation. Journal of Biological chemistry.270: pp.12607-12613.; goldman R (2004) Growth Factors and Chronic free health: panel, present, and future. Advances in Skin & round Care.17: pp.24-35.).

Chronic wounds are characterized by wounds that do not heal spontaneously within three months and occur in diabetic and vascular patients (Nunan R, harding KG, martin P (2014) Clinical questions of chronic grounds: search for an optimal animal model to treat the patient's complex. Diseases Models & mechanisms.7: 1205-1213.). The number of patients with Diabetic Foot Ulcers in chronic wounds is the largest, approximately 4 billion in the world, with 20% to 30% at risk of developing life-long Diabetic Foot Ulcers (Armstrong DG, boulton AJM, bus SA (2017) diabetes Foot Ulcers and the ir recurrence, new England Journal of medicine, 376: pp.2367-2375).

Chronic wounds are formed by molecular biological arrest in the inflammatory phase, not proceeding to the later proliferative phase, and thus abnormal gene expression and its interaction occur later (Bannon P, wood S, restivo T, campbell L, hardman MJ, mace KA (2013) Diabetes indexes stable in viral changes to fungal cells that are connected to a wound healing and healing dressing in semiconductor model & mechanisms.6: 1434-1447.).

It has been reported that, as The inflammatory phase continues, proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6) and Matrix Metalloproteinases (MMP) are expressed, in contrast to The decrease in The expression of various growth factors such as platelet-derived growth factor (PDGF), vascular Endothelial Growth Factor (VEGF) and insulin-like growth factor (IGF) (Trengove NJ, bielefeldt-Ohmann H, stable MC (2001) Mitogenic activity and cytokine peptides in non-neutral and neutral growth modules. Wound regeneration.8: pp.,13-25.; armstrong DG, jude EB (2002) of Matrix Metalloproteinases. 12. Journal of amino acids. 12. The present application is incorporated by reference.12.). Matrix Metalloproteinases (MMP), regulated by Matrix metalloproteinase inhibitor (TIMP), break down the extracellular Matrix and make re-epithelialization possible (Martins VL, caley M, O' Toole EA (2013) Matrix metal-localization and epidermal near repair. Cell and Tissue research.351: 255-268).

Among Matrix metalloproteinases, in particular, the study on Matrix metalloproteinase 9 (MMP-9) is the most active and is known to have the most adverse effect on chronic wounds (Jones JI, nguyen TT, peng Z, chang M (2019) Targeting MMP-9in diagnostic Foot Ulcers. Pharmaceuticals. 79.; reiss MJ, han YP, garcia E, goldberg M, yu H, garner WL (2010) Matrix metalloproteinase-delivery side dressing in person fashion. Surgery. 147-302).

Recently, the normal skin wound healing process has been problematic because chronic wounds caused by various causes of diabetic skin ulcer, bedsore, radiation ulcer, venous ulcer, cholesterol overuse, aging, etc. are difficult to be effectively treated. The recovery process of skin damage is inhibited by various causes, and abnormal inflammation delays progression to the proliferative phase in the inflammatory phase, which is the initial phase of wound recovery. Since the balance between matrix metalloproteinase activity and matrix metalloproteinase tissue inhibitory factor is broken, wound damage is sustained, synthesis/release of inflammatory cytokines such as tumor necrosis factor alpha is increased due to over-activated immune cells, expression of growth factors such as Transforming Growth Factor (TGF) beta is decreased, and progress to the proliferation stage is inhibited.

In particular, delayed wound healing of diabetic skin ulcer causes blood supply disorder, neuropathy, hepatitis, callus (callus) production, etc., which cause neovascularization, macrophage function, collagen proliferation, properties of granulation tissue, migration/activation of keratinocytes/fibroblasts, components of extracellular matrix, activity of matrix metalloproteinase, etc.

In the case of diabetic foot ulcers, about 15% of patients develop lesions, disorders, blockages of peripheral blood vessels, develop infections and eventually amputation of not only the wound site, but even the entire leg.

The existing treatments developed and put into practical use so far include various treatments such as blood sugar regulation, wound site removal, antibiotic therapy, and various forms of dressings.

Currently, effective Therapeutic methods or drugs for treating chronic wounds including Diabetic Foot ulcers are reported, and as understanding of skin wounds in terms of physiology, biochemistry, molecular biology, and the like has been advanced, various Therapeutic methods have been developed, for example, hyperbaric oxygen therapy for improving ischemia around wounds, skin transplantation, reduced pressure therapy, treatment of Endothelial Growth Factor (EGF), platelet Derived Growth Factor (PDGF), vascular Endothelial Growth Factor (VEGF), and the like, gene therapy, stem cell therapy, and the like (Kleopatra alexadou, john Doupis (2012) Management of diabetes foods, diabetes medicines, etc.; 3 (1) aurelio Pervila-Favila, margarta L Martinez-Fiere medicine, jessica G Rodriguez-Lazalde (2019rational Therapeutic residues), force 714, 4.

In particular, studies on growth factors for restoring diabetic skin ulcers, including insulin-like growth factor (IGF), transforming growth factor alpha, vascular endothelial growth factor, basic fibroblast growth factor (bFGF), platelet-derived growth factor, nerve Growth Factor (NGF), granulocyte-macrophage colony stimulating factor (GM-CSF), endothelial growth factor, hepatocyte Growth Factor (HGF), and the like, have been conducted (Grazul-Billska AT (2003) round healing: the role of growth factors. Drugs Today (Barc) 2003Oct 39 (10): 787-800.

According to the reportRecombinant Platelet Derived Growth Factor (PDGF) (local diffusion agent,

regranex corporation), vascular Endothelial Growth Factor (VEGF) (local diffusion agents,

genentech inc.) and the like, and the like. However, due to safety issues such as high cost, risk of causing cancer, etc., their use has been limited, and has recently been greatly reduced (McLaughlin PJ, cain JD, titunick MB, sassani JW, zagon IS. Focal Naltrexone Is a Safe and Effective Al-tertiary to Standard Treatment of metabolic woods.adv wooden Care.2017; 6.

Moreover, this growth factor treatment cannot effectively deliver matrix metalloprotease to the wound site, and requires multiple treatments at high concentrations, and the actual therapeutic effect is unstable. Moreover, in the case of recombinant growth factors, the ability to permeate the skin is low, and about 50 times the actual amount required for recovery in tissues is required, which causes a problem of high cost. Many studies have also been conducted for clinical application in Stem Cell therapy, which has recently received much attention as one of wound therapeutic agents (Lara Lopes, ocean Setia, afsha Aurshina, shirley Liu, haidi Hu (2018) Stem Cell therapy for diabetes spots emulsifiers: a review of clinical and clinical research. Stem Cell therapeutic: 9.

As one of the wound therapeutic agents, cephalexin (cephalexin) and clindamycin (clindamycin) are generally used as antibiotics, and in moderate or chronic infections, ampicillin (ampicillin) and imipenem (imipenem) are used.

Therefore, there is a need to develop drugs and cosmetics from natural resources, which have fewer side effects than the drugs currently used, are inexpensive and effective in treating and improving skin wounds or ulcers such as diabetic foot ulcers.

Arillus longan (Longanae arilus) is a seed coat of longan (Dimocarpus longan or Euphoria longan) or the same species belonging to Sapindaceae (Sapindaceae), and has been reported to contain glucose, fructose, protein and the like, and to have cardioprotective, appetite stimulating and the like effects (Chung b.s et al, dohaenyagyakdaajeon, you unggrimsa, 2nd ed.p. 197-198, 1998).

Ligusticum tenuissimum Kitagawa belongs to Umbelliferae (Umbelliferae), and is rhizome or root of Ligusticum sinense (Ligusticum sinense Oliv), ligusticum jensense (Ligusticum jenses Nakai et Kitagawa) or the same species, and it is reported that Ligusticum tenuissimum Rhizoma contains cnidilide (cnidilide), 3-butylphthalide (phthalide), and has antiviral effect (Chung B.S et al, dohae-hyangyakdaajeon, younggrimsa, 2nd Ed.P428-429, 1998).

Polygala tenuifolia Willd belonging to Polygalaceae (Polygalaceae) or Polygala tenuifolia Willd of the same species (Polygalae radix Polygalae) contain various saponins and are reported to have expectorant, antibacterial, etc. (Chung B.S. et al, dohaeanhygaakdaajeon, youngimsa, 2nd Ed.P798-799, 1998).

However, the above-mentioned documents cited as references in the present application do not disclose any report on the prevention or improvement activity of a combination crude drug extract of arillus longan, ligusticum sinensis and polygala tenuifolia which shows a potent therapeutic effect on skin wounds or ulcers such as diabetic foot ulcers by topical application.

Disclosure of Invention

Technical problem

In order to investigate the effect of a combined crude drug extract of arillus longan, ligusticum sinense, and polygala tenuifolia on the treatment or improvement of skin wounds or ulcers such as diabetic foot ulcers, the present inventors have intensively performed various experiments including: in vitro experiments such as an inhibitory effect on cytokine expression (in vitro) (experiment example 1), a cell growth promoting effect (in vitro) (experiment example 2), and a cell wound recovery effect (in vitro) (experiment example 3); and in vivo experiments such as therapeutic effects on chronic ulcers (in vivo) (example 4), wound healing effects (in vivo) (example 5), and inhibitory effects on expression of proinflammatory cytokines by growth factors (in vivo) (example 6). As a result of these investigations, the present inventors have confirmed that the combined crude drug extract of the present invention strongly inhibits and improves skin ulcer, and finally completed the present invention.

Technical scheme

In order to solve the problems of the background art, the present invention is directed to the development of a novel crude drug formulation for the treatment and prevention of skin ulcer.

Accordingly, an object of the present invention is to provide a topical pharmaceutical composition for treating and improving skin ulcer, comprising a combined crude drug extract of arillus longan, ligusticum sinense, and polygala tenuifolia as an active ingredient.

The term "combined crude drug extract" as defined in the present application means a combined crude drug extract which is (a) mixed at a mixing ratio of 0.01-100: 0.01-100, preferably at a mixing ratio of 0.1-50: 0.1-50 parts by weight (w/w), more preferably at a mixing ratio of 0.1-10: 0.1-10 parts by weight (w/w), even more preferably at a mixing ratio of 1-5: 1-5 parts by weight (w/w), most preferably at a mixing ratio of 1-3: 1-3 parts by weight (w/w), based on the dry weight (w/w) of arillus longan, ligusticum and polygala tenuifolia; or (b) the composition of the extracts of arillus longan, ligusticum sinense and polygala tenuifolia is in a mixing ratio of 0.01-100: 0.01-100 parts by weight (w/w), preferably in a mixing ratio of 0.1-50: 0.1-50 parts by weight (w/w), more preferably in a mixing ratio of 0.1-10: 0.1-10 parts by weight (w/w), even more preferably in a mixing ratio of 1-5: 1-5 parts by weight (w/w), most preferably in a mixing ratio of 1-3: 1-3 parts by weight (w/w), based on the dry weight (w/w) of arillus longan, ligusticum sinense and polygala tenuifolia.

It is still another object of the present invention to provide a thymic stromal lymphopoietin expression inhibitor comprising a combined crude drug extract of arillus longan, ligusticum sinense, and polygala tenuifolia as an active ingredient in an amount that inhibits Thymic Stromal Lymphopoietin (TSLP) cytokine.

The term "extract" as disclosed in the present application includes C which may be selected from water, e.g.methanol, ethanol, propanol, butanol etc ₁ -C ₄ Lower alcohol of (2), acetone, ethyl acetate, chloroform, hexane, butanediolThe extract extracted with one or more solvents selected from the group consisting of propylene glycol and glycerin preferably includes an extract extractable with one or more solvents selected from the group consisting of water and 10% to 90% (v/v) ethanol aqueous solution, and most preferably includes an extract extractable with one or more solvents selected from the group consisting of water and 20% to 80% (v/v) ethanol aqueous solution, but is not limited thereto.

The term "skin ulcer" disclosed in the present application includes, but is not limited to, bedsores, diabetic ulcers and the like.

Inflammation is part of a complex biological reaction of body tissues to harmful stimuli such as pathogens, injured cells, or irritants, and non-specific reactions such as fever, pain, redness, edema are referred to as "inflammatory reactions".

Inflammation is classified into the following categories: (a) Acute inflammation, the initial response of the body to noxious stimuli, is achieved by an increase in the movement of plasma and leukocytes (especially granulocytes) from the blood to the damaged tissue, and then by the spread of a series of biochemical phenomena and the maturation of the local vascular system, the immune system and the inflammatory responses associated with various cells within the damaged tissue; (b) Chronic inflammation, which is a long-term inflammation, is characterized by causing a progressive change in the cell type, such as monocytes, present at the site of inflammation, with simultaneous destruction and healing of the tissue occurring during the inflammation process.

Generally, macrophages of damaged cells release various cytokines to activate T lymphocytes, and mast cells as lymphocytes release various histidines to induce an internal barrier reaction to induce inflammation of infected cells. Thus, the expression level of cytokines can be used as an indicator that the inflammatory response is activated (otherwise, anti-inflammatory activity). The "anti-inflammatory activity" disclosed in the present application refers to an inhibitory activity against skin inflammation.

Cytokines refer to all immune substances including various stromal cells released by tumor necrosis factor produced by immune cells such as chemokines, interferons, interleukins, lymphokines and macrophages, B lymphocytes, T lymphocytes and mast cells, and cells in a broad sense including endothelial cells, and by immunology induced by invasion of fibroblasts and pathogens such as viruses.

Generally, cytokines are released only at the initial stage of infection, but are released continuously in the case where the immune system is abnormally activated. When cytokines are released at high levels for more than one week, the present inventors refer to "cytokine storm" (storm), which is a physiological response in which the innate immune system fails to regulate pro-inflammatory signaling molecules that become cytokines to cause excessive release, immune cells accumulate at the site of infection extremely abundantly to worsen inflammation, and the soothing of blood vessels causes extravascular bleeding, and even death, if severe. The term "inhibitory activity of cytokine expression" disclosed in the present application may be interpreted as preventing, treating or ameliorating a cytokine storm.

The term "cytokine" disclosed in the present application refers to various cytokines related to dermatitis such as atopic dermatitis, and specifically includes cytokines selected from the group consisting of thymic stromal lymphopoietin, colony Stimulating Factors (CSFs) such as granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), and Colony Stimulating Factors (CSFs) such as interleukin-1 (IL-1), IL-4, IL-10, IL-12, IL-13, IL-31, IL-33, and the like, tumor necrosis factor α (TNF- α), interferon γ (IFN γ), and the like, but is not limited thereto.

The extract of the present invention can be prepared by the following preferred embodiments.

In the case of the present invention, the above extract can be prepared in the following manner.

The term "combined crude drug extract of arillus longan, ligusticum sinense oliv and polygala tenuifolia" as defined in the present application can be prepared by the following steps: step 1: slicing arillus longan, rhizoma Ligustici, and cortex et radix Polygalae, and cleaning to obtain basic extract; based on the dry weight (w/w) of the longan pulp, the ligusticum and the polygala tenuifolia, the mixing ratio of 0.01-100: 0.01-100 parts by weight (w/w) in the step 2, preferably 0.1-50: 0.1-50 parts by weight (w/w), more preferablyOptionally mixing arillus longan, rhizoma Ligustici and cortex et radix Polygalae together at a mixing ratio of 0.1-10: 0.1-10 parts by weight (w/w), especially preferably at a mixing ratio of 1-5: 1-5 parts by weight (w/w), most preferably at a mixing ratio in the range of 1-3: 1-3 parts by weight (w/w) to obtain a mixed substance; step 3, adding C selected from water, such as methanol, ethanol, propanol, butanol, etc ₁ -C ₄ Preferably, an extraction solvent selected from the group consisting of water, methanol, ethanol, more preferably, an extraction solvent selected from the group consisting of water and 10 to 90% (v/v) aqueous ethanol, most preferably, an extraction solvent selected from the group consisting of water and 20 to 80% (v/v) aqueous ethanol is added to the mixed substance in a volume (v/w) of 1 to 20 times, preferably, in a volume (v/w) of 4 to 8 times; step 4, extracting each solution by hot water extraction, cold water extraction, reflux extraction or ultrasonic extraction at a temperature range of 50 ℃ to 120 ℃, preferably at a temperature range of about 80 ℃ to 100 ℃ for 1 hour to 24 hours, preferably for 2 hours to 12 hours, preferably by an extraction method of hot water extraction; and 5, repeating the extraction process, and drying each filtrate by filtering, freeze-drying or hot air drying, preferably collecting the combined crude drug extract of arillus longan, rhizoma Ligustici and cortex et radix Polygalae by freeze-drying each filtrate.

Still another object of the present invention is to provide a method for preparing a combined crude drug extract of arillus longan, ligusticum sinense oliver and polygala tenuifolia of the present invention as described above.

Another object of the present invention is to provide a pharmaceutical composition for topical application comprising the combined crude drug extract of arillus longan, ligusticum sinense, and polygala tenuifolia prepared by the above method as an active ingredient for treating and improving skin ulcer.

According to still another embodiment of the present invention, there is provided a method for treating and ameliorating skin ulcer in a mammal, comprising the step of topically administering to the mammal an effective amount of a combined crude drug extract of arillus longan, ligusticum sinense, and polygala tenuifolia, and a pharmaceutically acceptable carrier thereof.

In addition, according to another embodiment of the present invention, there is provided a use of a combined crude drug extract of arillus longan, ligusticum sinense, and polygala tenuifolia as an active ingredient for preparing a topical preparation for treating and improving skin ulcer in mammals including human beings.

The compositions of the invention may also contain conventional carriers, adjuvants or diluents depending on the method used. Preferably, the carrier is used as an appropriate substance according to the use and application method, but is not limited thereto. Suitable diluents are listed in the written text of Remington's Pharmaceutical Science (Mack Publishing co, easton PA).

The following preparation methods and excipients are merely examples, and are not intended to limit the present invention in any way.

The composition of the present invention may be provided as a topical pharmaceutical composition comprising a pharmaceutically acceptable carrier, adjuvant or diluent, and for example, may comprise lactose, sucrose, glucose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. The dosage form may also contain fillers, anticoagulants, lubricants, humectants, flavoring agents, emulsifiers, preservatives, and the like. The compositions of the present invention may be formulated by any of the procedures well known in the art for the rapid, sustained or delayed release of the active ingredient after administration to a patient.

For topical administration, the extract of the present invention may be formulated in the form of ointments and creams, including creams, gels, masks, spray solutions, emulsions, ointments, lotions, liniments (liniments), balms, solutions, suspensions, cosmetic sheets, patches, sprays, cataplasms, and like topical preparations.

The compositions of the invention in the form of their pharmaceutically acceptable salts can be used in the form of their pharmaceutically administrable forms, not only alone or in suitable combination, but also in combination with pharmaceutically active compounds.

The preferred dosage of the extract or composition of the present invention varies depending on the condition and body weight of a subject, the severity of a disease, the form of a drug, the route and period of administration, and can be selected by the relevant practitioner. However, in order to obtain the preferred effect, it is generally recommended to administer the extract of the invention topically in an amount of 0.01g/kg-10g/kg, preferably in an amount ranging from 1g/kg to 5g/kg (weight/day). The dose can be administered in one dose or in multiple doses per day. From the viewpoint of the composition, the present extract is contained in an amount of 0.01 to 80% by weight, preferably 0.5 to 50% by weight, based on the total weight of the composition.

The present inventors confirmed that the combined extract of the present invention is very useful for the improvement or treatment of skin ulcer in the form of a topical pharmaceutical or cosmetic composition by performing in vitro experiments on the inhibitory effect on cytokine expression (in vitro) (experimental example 1), the cell growth promoting effect (in vitro) (experimental example 2), the cell wound healing effect (in vitro) (experimental example 3), and the like, and in vivo experiments on the therapeutic effect on chronic ulcer (in vivo) (experimental example 4), the wound healing effect (in vivo) (experimental example 5), the inhibitory effect on the expression of proinflammatory cytokines of growth factors (in vivo) (experimental example 6), and the like.

It is still another object of the present invention to provide a cosmetic composition comprising a combined crude drug extract of arillus longan, ligusticum sinense oliv, and polygala tenuifolia as an active ingredient in an amount effective for treating and improving skin ulcer.

The cosmetic composition of the present invention comprises 0.001 weight percent to 40 weight percent of the composition of the present invention, preferably 0.01 weight percent to 10 weight percent of the composition of the present invention, based on the total weight of the composition. The other ingredients may be a mixture of ingredients of a general cosmetic composition well known in the art to which the present invention pertains.

The formulation of the cosmetic comprising the above composition may be prepared in any form such as a skin lotion, a skin softening lotion, a skin toning lotion, an astringent, a skin lotion, a milky lotion, a moisturizing lotion, a nourishing lotion, a massage cream, a nourishing cream, a moisturizing cream, a hand cream, a foundation, an essence, a nourishing essence, a mask, a cleansing foam, a cleansing lotion, a makeup removing cream, a body lotion, a body wash, a hair cream, a beauty lotion, and the like.

The following preparation methods and excipients are merely examples, and are not intended to limit the present invention in any way.

The cosmetic composition of the present invention may further comprise additional additives selected from the group consisting of water-soluble vitamins, fat-soluble vitamins, peptide polymers, polysaccharide polymers, sphingolipids and seaweed extracts.

Preferably, the water-soluble vitamin may be mixed with a cosmetic, preferably, the multivitamin may be, for example, vitamin B ₁ 、B ₂ Vitamin B ₆ Pyridoxine, pyridoxine hydrochloride, vitamin B ₁₂ Pantothenic acid, nicotinic acid, nicotinamide, folic acid, vitamin C, vitamin H, and the like, salts thereof such as vitamin hydrochloride, sodium ascorbate, and the like, or derivatives thereof such as sodium ascorbate-2-phosphonate, and magnesium ascorbate-2-phosphonate, and these vitamins can be obtained by a conventional method such as a microbial conversion method, a method of purification from a microbial culture, a biological enzymatic method, or a chemical synthesis method.

The preferred fat-soluble vitamins may be combined with cosmetic products, and preferably, the fat-soluble vitamins used in embodiments of the present invention include multivitamins, such as vitamin A, vitamin D ₂ Vitamin D ₃ Vitamin E (dl-a-tocopherol, D-D-tocopherol) and derivatives thereof, such as ascorbyl palmitate, ascorbyl stearate, ascorbyl dipalmitate, acetic acid-dl-a-tocopherol, nicotinic acid dl-a-tocopherol vitamin E, dl-panthenol, D-panthenol, pantothenyl ethyl ether and the like, can be obtained by a general method such as a microbial conversion method, a method of purification from a microorganism, a biological enzyme method, a chemical synthesis method and the like.

Preferred peptide polymers may be mixed with cosmetics, and preferably, the peptide polymers used in the embodiments of the present invention include collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed gel, keratin, and the like.

The polysaccharide polymer is preferably hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate, or a salt thereof (Na such as sodium salt). For example, chondroitin sulfate can be purified from mammals or fishes and used.

Preferred sphingolipids may be mixed with cosmetics, and the sphingolipids are preferably ceramide, phytosphingosine, sphingolipopolysaccharide, and the like. Sphingolipids can be obtained from mammals, fish, shellfish, yeast, or plants by a conventional method.

The preferred seaweed extract (seaweed extract) may be mixed with cosmetic, and is preferably an extract of brown algae, red dates, green algae, etc., or carrageenan, alginic acid, sodium alginate, potassium alginate, etc. isolated and purified from them. The seaweed extract (algae extract) is obtained by purifying seaweed.

The cosmetic composition of the present invention may be prepared by combining the above-mentioned essential components with other components used in a usual cosmetic composition, as required.

Preferably, the other ingredients described above may include oil ingredients, moisturizers, softeners, surfactants, organic or inorganic dyes, organic powders, ultraviolet absorbers, preservatives, antioxidants, plant extracts, pH regulators, alcohols, pigments, perfumes, refrigerants, blood circulation improvers (circulators), antiperspirants (antiperspirants), distilled water, and the like.

Preferably, the oil component may include ester oil, hydrocarbon oil, silicone oil, fluoride oil, animal oil, vegetable oil, and the like.

<xnotran> , -2- , -2- , , , , , , , , , , , , , , , , , , , , , -2- , , -2- , , , , , , , , , , , , , , , , , , , 2- , 2- , , , , , , , , , , , , , , , , , </xnotran> Cetyl neodecanoic acid, octyldodecyl neodecanoic acid, isohexadecyl isostearic acid, isostearyl isostearic acid, octyldecyl isostearic acid, polyglycerol oleanolic acid ester, polyglycerol isostearate, triisohexadecyl citric acid, triisoalkyl citric acid, triisooctyl citric acid, lauryl lactic acid, myristyl lactic acid, cetyl lactic acid, octyldecyl lactic acid, triethyl citrate, acetyl tributyl citrate, trioctyl citric acid, diisostearyl maleic acid, di-2-ethylhexyl hydroxystearic acid, 2-ethylhexyl succinic acid, diisobutyl adipic acid, diisopropyl sebacate, dioctyl sebacate, cholesterol stearic acid, cholesterol isostearic acid, cholesterol hydroxystearic acid, cholesterol oleic acid, dihydrocholesterol oleic acid, phytosterol isostearic acid, phytosterol oleic acid, isohexadecyl 12-stearoyl hydroxystearic acid, stearyl 12-stearoyl hydroxystearic acid, isostearyl 12-stearoyl hydroxystearic acid.

Preferably, the hydrocarbon oil described above includes squalene, liquid paraffin, α -olefin oligomer, isoparaffin, ceresin, paraffin, liquid isoparaffin, polybutene, microcrystalline wax, and vaseline.

The silicone oil described above may preferably include polymethylsilicone oil, methylphenylsiloxane, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane-methylhexadecyloxysiloxane copolymer, dimethylsiloxane-methyloctadecyloxysiloxane copolymer, alkyl-modified silicone oil, amino-modified silicone oil, and the like.

Preferably, the fluorochemical oil described above may comprise perfluoropolyether and the like.

Preferably, the animal or vegetable oil may include avocado oil, almond oil, olive oil, sesame oil, rice bran oil, safflower oil, soybean oil, corn oil, rapeseed oil, bitter almond oil, palm kernel oil, palm oil, castor oil, sunflower oil, fruit seed oil, cottonseed oil, coconut oil (cocout palm oil), kukukui nut oil (cucuii nut oil), wheat germ oil (wheat germ oil), rice germ oil, shea butter, evening primrose oil, macadamia nut oil (marker damyma nut oil), meadowfoam seed oil (medo home oil), egg yolk oil, lanolin, hemp seed oil, mink oil, sweet orange oil (orange roupy oil), jojoba oil, hemp wax, liquid lanolin, solid castor wax, and the like.

Preferably, the moisturizer may include a water-soluble low molecular moisturizer, a lipophilic low molecular moisturizer, a water-soluble polymer, a fat-soluble polymer, and the like.

Specifically, preferred water-soluble low-molecular moisturizers may include serine, glutamine, sorbitol, mannitol, sodium pyrrolidone carboxylate, glycerin, propylene glycol, 1, 3-butylene glycol, ethylene glycol, polyethylene glycol (degree of polymerization > 2), polypropylene glycol (degree of polymerization > 2), lactic acid, lactate, and the like.

Preferred fat-soluble low molecular moisturizers may include cholesterol, cholesterol ester, and the like.

Preferred water-soluble polymers may include carboxyethyl polymers, polyaspartate, tragacanth, xanthan gum, hydroxymethylcellulose (HMC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), carboxymethylcellulose, water-soluble chitin, chitosan, dextrin, and the like.

Preferred liposoluble polymers may include polyvinylpyrrolidone-eicosene copolymer, polyvinylpyrrolidone-hexadecene copolymer, cellulose nitrate, dextrin fatty acid esters, silicone polymers, and the like.

Preferred emollients may include cholesterol esters of long chain acyl glutamic acid, cholesteryl hydroxystearic acid, 12-hydroxystearic acid, rosin acid (rogec acid), cholesterol esters of lanolin fatty acids, and the like.

Preferred surfactants may include nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, and the like.

Specifically, preferred nonionic surfactants may include self-emulsifying glyceryl monostearate, propylene glycol fatty acid ester, glyceryl local fatty acid ester, sorbitan fatty acid ester, polyoxyethylene (POE) sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene fatty acid ester, polyoxyethylene solid castor oil, polyoxyethylene-polyolefin plastomer (POE-POP) copolymer, polyoxyethylene-polyolefin plastomer alkyl ether, polyether modified silicone, lauryl alkanolamide, alkyl amine oxide, hydrogenated soybean phospholipid, and the like.

Preferred anionic surfactants may include fatty acid soaps, alpha-acyl sulfonates, alkyl sulfonates, alkylaryl sulfonates, alkylnaphthalene sulfonates, alkyl sulfonates, polyoxyethylene alkyl ether sulfates, alkylamide sulfates, alkyl phosphates, polyoxyethylene alkyl phosphates, alkylamide phosphates, alkanoyl alkyl taurates, N-acyl-amino acid salts, polyoxyethylene alkyl ether carboxylates, alkyl sulfosuccinates, alkyl sulfoacetates, acylated hydrolyzable collagen peptide salts, perfluoroalkyl phosphate esters, and the like.

Preferred cationic surfactants may include: alkyltrimethyl ammonium chloride, stearyl trimethyl ammonium bromide, cetearyltrimethyl ammonium chloride, distearyldimethyl ammonium chloride, stearyl dimethylbenzyl ammonium chloride, behenyltrimethyl ammonium bromide, benzalkonium chloride, diethylaminoethyl stearate, dimethylaminopropionamide stearate, lanolin derivative quaternary ammonium salts, and the like.

Preferred amphoteric surfactants may include carboxybetaine type, amidobetaine type, hydroxysulfobetaine type, phosphobetaine type, aminocarboxylic acid, imidazoline derivative type, amidoamine type, and the like.

Preferred organic and inorganic dyes may include: inorganic dyes, silicic acid, silicic anhydride, magnesium silicate, talc, sericite, mica, kaolin, mengla, crolein, bentonite, titanium film mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, aluminum oxide, calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, ferrous oxide, chromium hydroxide, calamine, carbon black and their compounds; organic dyes, polyamides, polyesters, polypropylenes, polystyrenes, polyurethanes, vinyl resins, urea resins, phenolic resins, fluororesins, silicone resins, acrylic resins, melamine resins, epoxy resins, polycarbonate resins, divinylbenzene-styrene copolymers, fibroin, cellulose, CI pigment yellow, CI pigment orange, and composites thereof.

Preferred organic powders may include: metal soaps, such as calcium theate; metal alkyl phosphates (alkyl phosphate metal salts), such as sodium cetyl phosphate, zinc laurate, calcium laurate; polyvalent metal acylamino acid salts such as calcium N-lauroyl-b-alanine, zinc N-lauroyl-b-alanine, calcium N-lauroyl-glycinate, etc.; polyvalent metal amide sulfonate salts such as calcium N-lauroyl taurate, calcium N-palmitoyl taurate and the like; n-acyl basic amino acids such as N epsilon-lauroyl-L-lysine, N epsilon-palmitoyl lysine, N alpha-palmitoyl ornithine, N alpha-lauroyl arginine, hydrogenated lanolin fatty acid acyl arginine, etc.; n-acyl polypeptides, such as N-lauroyl glycine; α -amino fatty acids such as α -aminocaprylic acid, α -aminolauric acid, and the like; and polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, divinylbenzene-styrene copolymer, tetrafluoroethylene, etc.

Preferred ultraviolet absorbers may include p-aminobenzoic acid, ethyl p-aminobenzoate, amyl p-aminobenzoate, octyl p-aminobenzoate, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, homomenthyl salicylate, benzyl cinnamate, p-methoxycinnamate-2-ethoxyethyl, octyl p-methoxycinnamate, mono-2-ethylglycerol di-p-methoxycinnamate, isopropyl p-methoxycinnamate, a diisopropyl-diisopropyl cinnamate mixture, urocanic acid, ethyl urethanoate, hydroxymethoxybenzophenone sulfonic acid and salts thereof, dihydroxymethoxybenzophenone disulfonate (dihydromethoxy benzophenone disulphonate Na), dihydroxybenzophenone, tetrahydroxybenzophenone, 4-tert-butyl-4 ' -methoxybenzoylmethane, 2,4, 6-trianilino-p- (carboxy-2 ' -ethylhexyl-1 ' -oxy) -1,3, 5-triazine, 2- (2-hydroxy-5-methylphenyl) benzotriazole, and the like.

Preferred preservatives may include hinokitiol, trihydrochloric acid, triclosan, chlorhexidine gluconate, phenoxyethanol, resorcinol, isopropyl methylphenol, azulene, salicylic acid, lithium zinc sulfide, bexanium chloride hydrochloride, sensitizer 301, sodium mononitroguaiacol, undecylenic acid, and the like.

Preferred antioxidants may include butyl hydroxyanisole, propyl gallate, edetic acid, and the like.

Preferred pH adjusting agents may include citric acid, sodium citrate, malic acid, sodium malate, fumaric acid, sodium fumarate, succinic acid, sodium succinate, sodium hydrogen phosphate, and the like.

Preferred alcohols may include cetyl alcohol and the like.

Also, other ingredients that may be added to the above ingredients and the amounts thereof are not limited within the scope of the objects and effects of the present invention, and preferably, the content of the other ingredients is in the range of 0.01% to 5%, more preferably, 0.01% to 3% of the total content of the composition.

The cosmetic composition of the present invention may be modified into a solution, emulsion, adhesive mixture, and the like.

The above-mentioned components such as water-soluble vitamins, fat-soluble vitamins, peptide polymers, polysaccharide polymers, sphingolipids, seaweed extracts, and additional components other than the above-mentioned components, which may be added as required, can be obtained by a conventional method described in the literature (Matsumoto Mithio; manual for the definition of transdermal applied preparation. Seisi Press,1st Ed., 1985).

The compound of the present invention has no toxic side effect and thus can be used safely.

It will be apparent to those skilled in the art that various modifications and variations can be made in the composition, use and formulation of the present invention without departing from the spirit or scope of the invention.

The present invention is more specifically illustrated by the following examples. However, the present invention should not be construed as being limited to these examples in any way.

Advantageous effects

As described above, the present inventors have focused on inflammation in vitro experiments such as an inhibitory effect on cytokine expression (in vitro) (experimental example 1), a cell growth promoting effect (in vitro) (experimental example 2), and a cell wound recovery effect (in vitro) (experimental example 3), and in vivo experiments such as a therapeutic effect on chronic ulcers (in vivo) (experimental example 4), a wound recovery effect (in vivo) (experimental example 5), and an inhibitory effect on proinflammatory cytokine expression of growth factors (in vivo) (experimental example 6), and have focused on the potent therapeutic effect on skin ulcers. Therefore, it was confirmed that the combined extract of the present invention is very useful in the improvement or treatment of skin ulcer in the form of a topical pharmaceutical agent or cosmetic composition.

Drawings

FIG. 1 shows a photograph of cells of a test group treated with the combined extract of the present invention after wound induction by skin epithelial cells (HaCaT) (DIW: distilled water; NP: the combined extract of the present invention).

FIG. 2 shows establishment of streptozotocin (STZ: streptozotocin) -induced diabetes mouse model (STZ: streptozotocin, DIW: distilled water; NP: the combined extract of the present invention).

FIG. 3 shows the healing effect of the combined extract (NP) of the present invention on the healing of diabetic skin wounds, as days elapsed after the injury (Con: control group; STZ: streptozotocin, DIW: distilled water; NP: combined extract of the present invention).

Fig. 4 shows histological analysis of diabetic skin wounds and the promoting effect of the combined extract (NP) of the present invention on the proliferation of granulation tissue (scale bar =200 μm, STZ: streptozotocin, DIW: distilled water; NP: the combined extract of the present invention).

Detailed Description

It will be apparent to those skilled in the art that various modifications and variations can be made in the composition, use and formulation of the present invention without departing from the spirit or scope of the invention.

The present invention is more specifically illustrated by the following examples.

However, the present invention should not be construed as being limited to these examples in any way.

Examples

Without limiting the scope of the present invention, the following examples and experimental examples are intended to additionally illustrate the present invention.

Example 1 preparation of the Combined extract of the invention (1)

After finely cutting 20g of dried longan pulp (Buyoung Yakup Co. Ltd.), 20g of dried Ligusticum sinense (Buyoung Yakup Co. Ltd.), and 20g of dried Polygala tenuifolia (Buyoung Yakup Co. Ltd.) were mixed with 6 times by volume (v/w) of a 20% aqueous ethanol solution, the mixture was extracted under reflux at 90. + -. 5 ℃ for 3 days. After removing the residue by filtering the extract through filter paper (pore size: less than 10 μm), the remaining residue was re-extracted twice using 4 volumes (v/w) of 20% aqueous ethanol solution, and then the extract was filtered through filter paper (pore size: less than 10 μm).

The collected extracts were mixed together and concentrated under vacuum (16-21 brix) to obtain a concentrated extract. After the concentrated extract was dried by the freeze-drying process, 20.5g of the combined extract (1) of the present invention (hereinafter, referred to as "WIN-1001X") was obtained by pulverization (less than 50 mesh) (dried powder, yield 33.4%).

Examples 2 to 6 preparation of the Combined extract of the invention (2) to the Combined extract of the invention (6)

A plurality of inventive combined extracts of arillus Longan (LA), ligusticum (LT), and polygala tenuifolia (PR), i.e., the inventive combined extract (2) to the inventive combined extract (6), were obtained in the same procedure as in example 1 except that different mixing ratios and different solvents were used, and they were used as test samples in the following experiments.

TABLE 1

Various combined extracts

Experimental example 1 inhibitory Effect on cytokine expression (in vitro).

To determine the anti-inflammatory activity of the extracts of the invention, the following cytokine expression inhibition assay using HaCaT cells was performed according to the procedures described in the literature (Jeong et al, 2019, J.invest.Dermatol., may;139 (5): pp 1098-1109).

HaCaT cells (human epithelial keratinocytes, 300493, CLS) were inoculated in Du's modified Igor medium (DMEM) containing 10% fetal bovine serum, 100 units/ml penicillin, 100. Mu.g/ml streptomycin (D6429, sigma-Aldrich Co. Ltd.), while maintaining optimal humidity (85% -95%) and 5% CO ₂ In an incubator (HERA c) under atmospheric conditionsell 150i, thermo Fisher Scientific co.ltd.).

For gene expression assay, cultured cells were transferred to 12 wells and treated with tumor necrosis factor α (RC 214-12, biobasic Co. Ltd.) at 50ng/ml for 1 hour to induce inflammatory response. Dexamethasone (200 nM, positive control, "DEX", D4902, sigma-Aldrich co.ltd. Company) and distilled water (negative control, "DIW") were used as comparative controls.

1 hour after the induction of inflammation, 1. Mu.g/ml of the extract of the present invention prepared in example was treated with the same medium and cultured for 1 hour. After the culture, ribonucleic acid (RNA) was extracted from the cells (FATRR-001, favorgen Co.), and then complementary deoxyribonucleic acid (cDNA) was synthesized from the RNA using a complementary deoxyribonucleic acid (cDNA) synthesis kit (RRO 36A, TAKARA Co.). After polymerization using the synthesized complementary deoxyribonucleic acid and Sybrgreen kit (RT 500M, enzynomics), real-time Polymerase Chain Reaction (PCR) was performed using primers (RPLPO, TSLP, GM-CSF, and IL-1. Beta.) related to various cytokines involved in skin inflammation, as shown in Table 2.

TABLE 2

Primers for use in real-time polymerase chain reaction (RT-PCR) methods

The quantitative results of the real-time polymerase chain reaction are shown in table 3, and it can be seen from table 3 that the test sample group treated with the extract of the present invention rapidly inhibited the expression levels of various cytokines associated with skin inflammation compared to the negative control group treated with distilled water (DIW), and it was confirmed that the inhibitory activity of the test sample against the expression of various cytokines associated with skin inflammation was the same as that of the positive control group treated with Dexamethasone (DEX).

Therefore, it was confirmed that each of the combined extracts of the present invention prepared in examples 1 to 6 had a strong inhibitory effect on skin inflammation.

TABLE 3

Inhibitory Effect on cytokine expression

Experimental example 2 cell proliferation promoting Effect (in vitro).

To determine the promoting activity of the extracts of the invention, the following cell proliferation assay using HaCaT cells was performed according to the procedures described in the literature (Lee et al, 2020, int J Mol Sci.2020Jan 5 (1): 343.

HaCaT cells (human epithelial keratinocytes, 300493, CLS), cerebral capillary endothelial cells (bEND. 3, CRL-2299 ATCC) and fibroblasts (NIH 3T3, CRL-1658 ATCC) were inoculated in Du's modified Igor medium (D6429, sigma-Aldrich Co. Ltd.) containing 10% fetal bovine serum, 100 units/ml penicillin, 100. Mu.g/ml streptomycin (D6429, sigma-Aldrich Co. Ltd.), and 100. Mu.g/ml streptomycin (D6429, sigma-Aldrich Co. Ltd.), while maintaining optimum humidity (85% -95%) and 5% CO ₂ The culture was carried out in an incubator (HERA cell 150i, thermo Fisher Scientific Co. Ltd. Co.) under atmospheric conditions.

To determine the cell proliferation promoting effect of the extract of the present invention, the cultured cells were transferred to 48 wells, and the extract of the present invention prepared in example of 1. Mu.g/ml and distilled water were treated separately (negative control group, "DIW").

The cells were treated with 10. Mu.L/ml of Quanti-MaxTM (QM 1000, BIOMAX) repeatedly at intervals of 0 hour, 24 hours, 48 hours and 72 hours, respectively, and cultured for 30 minutes. After the incubation, the absorbance of each group was measured at a wavelength of 450nm using a microplate reader (SPECTRA MAX 250, molecular Devices), and the test results are shown in Table 4 below.

As can be seen from table 4 below, it was confirmed that the combined extracts of the present invention prepared in examples 1 to 6 had a potent cell growth promoting effect.

TABLE 4

Cell proliferation promoting effect

Experimental example 3 cellular wound recovery effect (in vitro).

To determine the restorative activity of the extract of the invention, the following cell wound test using HaCaT cells was performed according to literature procedures (Na et al, 2016, J Invest Dermatol.2016Apr 136 (4): 847-858).

HaCaT cells (human epithelial keratinocytes, 300493, CLS) were seeded in Du's modified Igor medium (D6429, sigma-Aldrich Co. Ltd.) containing 10% fetal bovine serum, 100 units/ml penicillin, 100. Mu.g/ml streptomycin, while maintaining optimum humidity (85% -95%) and 5% CO ₂ The culture was carried out in an incubator (HERA cell 150i, thermo Fisher Scientific Co. Ltd. Co.) under atmospheric conditions.

To determine the cell wound recovery effect of the extract of the present invention, cultured cells were transferred to 6 wells and cultured until the cell fusion of the medium reached about 90%. The cultured cells were cultured in serum-free medium (D6429, sigma-Aldrich) for 24 hours. After the cultured cells were scratched using 200. Mu.l of Tip (KG 1212-L, kirgen), the medium was transferred to a new 2% fetal bovine serum medium (D6429, sigma-Aldrich Co.) containing 1. Mu.g/ml of the extract of the present invention prepared in example. Photographs of the recovery process were taken using a microscope (AMEX 1000, EVOS XL Core) and compared at different times, and distilled water (negative control, "DIW") was used as a negative control.

As can be seen from table 5 below, it was confirmed that the combined extracts of the present invention prepared in examples 1 to 6 had potent cell wound recovery effects.

TABLE 5

Cell wound recovery effect

Experimental example 4 therapeutic effects on chronic ulcer (in vivo).

To confirm the therapeutic effect of the extract of the present invention on chronic ulcers, an animal model test using mice was performed according to the reference (Long M, rojo de la Vega M, wen Q, bharara M, jiang T, zhang R, zhou S, wong PK, wondrak GT, zheng H, zhang DD (2016) An antisense Role of NRF2in Diabetic Wound Healing. Diabetes 65: 780-793).

4-1. Preparation of diabetic mouse model

C57BL male mice (230g, daehanbiolink Co. Ltd.) of 8 weeks age were raised in a raising room maintained at 22. + -. 1 ℃ and 50. + -. 5% respectively with light irradiation adjusted at 12-hour intervals.

After 1 week of acclimation, the mice were divided into test sample groups and control groups (1 mouse per feeding room). 50mg/kg of streptozotocin (S0130, STZ, sigma-Aldrich, USA (USA)) was intraperitoneally administered to the test specimen groups for 5 consecutive days, and 0.05M sodium citrate buffer (pH 4.5, IBS-BC0036, intron, korea) was intraperitoneally administered to the negative control group for 5 consecutive days.

3 weeks after administration, blood samples were taken every 4 hours to measure fasting plasma glucose resin in the tail of the mice, and only mice with fasting plasma glucose above 350mg/dl were selected in the experiment.

50mg/kg of streptozotocin (S0130, STZ, sigma-Aldrich Co., USA (USA)) was intraperitoneally administered to the test sample groups for 5 consecutive days (refer to FIG. 2), and 0.05M sodium citrate buffer solution (pH 4.5, IBS-BC0036, intron Co., korea (Korea)) was intraperitoneally administered to the negative control group for 5 consecutive days. Fasting blood glucose values were measured for 4 hours and mouse body weights at weeks 3 and 5.

As is clear from tables 6 and 7, it was confirmed that STZ-diabetes-induced mice had reduced body weight and increased fasting plasma glucose values. Selected mice with fasting blood glucose values greater than 350mg/dL were considered the diabetes-model group (Long M, rojo de la Vega M, wen Q, bharara M, jiang T, zhang R, zhou S, wong PK, wondrak GT, zheng H, zhang DD (2016) An antisense Role of NRF2in Diabetic Wound Healing.diabetes.65: 780-793).

TABLE 6

STZ-induced weight changes in diabetes-model mice

TABLE 7

STZ-induced glycaemic changes in diabetes-model mice

4-2. Experimental methods (therapeutic effect on chronic ulcer)

To confirm the therapeutic effect of the extract of the present invention on chronic ulcers, animal model tests using diabetes-induced mice prepared in the above-described step 4-1 were performed according to the methods described in the references (Long M, rojo de la Vega M, wen Q, bharara M, jiang T, zhang R, zhou S, wong PK, wondrak GT, zheng H, zhang DD (2016) An established roll of NRF2in diabetes bound Healing. Diabetes.65: 780-793).

C57BL male mice (230g, daehanbiolink Co. Ltd.) aged for 8 weeks were divided into (a) a control group and (b) a diabetes-induced mouse group prepared in the above step 4-1, and the mice were anesthetized by intraperitoneal injection of 300. Mu.l of tribromoethanol (Avertin) (25 mg/mL T48402, sigma-Aldrich Co., USA).

After confirmation of anesthesia, the backs of the mice were shaved using an electric razor (327/808, RIKEI, taiwan, china) and a 5mm diameter circular whole dorsal skin layer was cut using a 5mm biopsy punch (BP-50F, kai Industries, inc., USA) and surgical scissors (PF-24.10, professional, pakistan). Skin wounds of the test sample group were coated with 35. Mu.l of the extract of the present invention dissolved in distilled water (10 mg/mL) per day, and skin wounds of the negative control group were coated with 35. Mu.l of distilled water per day.

The skin wounds were photographed and imaged using a digital camera (LD V20 digital camera) for 14 days, and the size of the skin wounds was measured in a quantitative manner using Photoshop CS5 program (Adobe corporation).

The skin wound healing rate was calculated by dividing each sample by the size of the wound on day 0 according to the following mathematical formula 1.

Mathematical formula 1

Skin wound cure rate = (nth day/0 th day) × 100

4-3. Test results (skin ulcer)

As can be seen from fig. 3, the cure rate of the diabetes-induced group was decreased as compared with the negative control group treated with distilled water, whereas the cure rate of the test sample group was significantly increased as compared with the diabetes-induced group and the negative control group.

After the wound site was calculated by photographing the wound site using a camera (LG V20 cell phone camera), comparison was performed in a quantitative manner by calculating pixels. As can be seen from table 8, the combined extract of the present invention was confirmed to exhibit a faster healing effect at the skin ulcer sites in diabetic mice starting on day 6 after the skin injury, as compared to the negative control group.

TABLE 8

Improving effect on skin ulcer area

EXAMPLE 5 wound healing efficacy (in vivo).

To confirm the Wound-healing effect of the extract of the present invention, the diabetes-induced mice prepared in the above step 4-1 were used as subjects, and histological differences of the test sample group and the control group were compared according to the method disclosed in the reference and based on the test results of step 4-3 (Long M, rojo de la Vega M, wen Q, bharara M, jiang T, zhang R, zhou S, wong PK, wondrak GT, zheng H, zhang DD (2016) Anes sensory Role of NRF2in diabetes bound Healing. Diabetes 65: 780-793).

5-1. Experimental methods (wound recovery Effect)

C57BL male mice (230g, daehanbiolink Co. Ltd.) aged for 8 weeks were divided into (a) a control group and (b) a diabetes-induced mouse group prepared in the above step 4-1, and the mice were anesthetized by intraperitoneal injection of 300. Mu.l of tribromoethanol (Avertin) (25 mg/mL T48402, sigma-Aldrich Co., USA).

After confirmation of anesthesia, the mice were shaved on their backs using an electric razor (327/808, RIKEI, taiwan, china), and a 5mm diameter circular, monolithic dorsal skin layer was cut using a 5mm biopsy punch (BP-50F, kai Industries, USA) and surgical scissors (PF-24.10, professional, pakistan). Skin wounds of the test sample group were coated with 35. Mu.l of the extract of the present invention dissolved in distilled water (10 mg/mL) per day, and skin wounds of the negative control group were coated with 35. Mu.l of distilled water per day.

On day 7 of skin injury, wound tissue was surgically isolated, and the isolated wound tissue was soaked with 4% paraformaldehyde solution (158127, sigma, usa) and fixed by stirring overnight in a shaker (4 ℃).

The following day, wound tissue was placed in vials containing Phosphate Buffered Saline (PBS) and washed 5 times at Room Temperature (RT) 15 minutes apart.

Then, the wound tissue was dehydrated by placing it in 25%, 50%, 75%, 95% and 100% ethanol solutions in this order on a shaker (SHK 039, jeong Biotech, korea) for 30 minutes.

The dehydrated skin tissue was transferred to a xylene solution (1330-20-7, DUKSAN, korea (Korea)) and left in a vacuum cleaner for 2 hours to allow xylene to permeate into the tissue.

After confirming the transparency of the tissue, the tissue containing xylene was transferred to an incubator (Oven 300, CHICAGO SURGICAL &ELECTRICAL CO., USA) at 55 ℃ and washed 5 times with a paraffin solution (8042-47-5, merck Millipore, germany). After the final addition of the paraffin solution, the temperature of 55 ℃ was maintained in the incubator overnight. The following day, the tissue was embedded in paraffin and left to stand at 4 ℃ for one day after 1 hour of curing at Room Temperature (RT). The tissue embedded in paraffin was cut to a thickness of 5 μm using a microtome (820, AO AMERICAN OPTICAL, U.S.A.), and the section was placed on a slide glass. The remaining paraffin was removed using xylene, and the tissues were hydrated sequentially with 100%, 95%, 75%, 50% and 25% ethanol solutions. Tissues were then stained with hematoxylin and eosin (H & E), photographed and analyzed using an EVOS XL Core microscope (usa, magnification 40).

4-3 test results (skin wound)

As can be seen from fig. 4, it is interesting that granulation tissue not observed in the control group was observed in the experimental sample group treated with the combined extract of the present invention. Granulation tissue is composed of many blood new vessels, fibroblasts, and cells such as growth factors, which are formed during the proliferation process of wound healing (Grotendorst GR, martin GR, pencv D, sodek J, harvey AK (1985) Stimulation of growth tissue by tissue formation-derived growth factor in normal and biological rates, the clinical of clinical information 76: 2323-2329.).

The frequency of observation of granulation tissue was 5 times or more higher in the test sample group than in the control group (see table 9).

TABLE 9

Recovery effect on skin wound

	Granulation tissue formation (%)
		STZ+DIW	14
STZ+NP	71.4

Experimental example 6 inhibitory Effect (in vivo) on expression of proinflammatory cytokines in relation to growth factors.

To confirm the inhibitory effect of the extracts of the present invention on the expression of growth factor-related proinflammatory cytokines, the following cytokine expression inhibition assays using experimental animals were performed according to procedures disclosed in the literature (Long M, rojo de la Vega M, wen Q, bharara M, jiang T, zhang R, zhou S, wong PK, wondrak GT, zheng H, zhang DD (2016) and Essential roll of NRF2in diabetes bound Healing. Diabetes 65: 780-793.).

It has been reported that, in chronic wounds, the abnormal expression of Matrix metalloproteinase-9 is increased and The expression of growth factors (platelet-derived growth factor, vascular endothelial growth factor, etc.) is decreased (Trengove NJ, bielefeldt-Ohmann H, standard MC (2001) genetic activity and cytokine levels in non-genetic and genetic leg effectors. Round Repair and regeneration.8:13-25.; armstronG, jude EB (2002) The roll of Matrix Metalloproteinases in round heating. Journal of The American great Medical association.92: 12-18.).

In particular, growth factors promote the formation of granulation tissue that mediates Wound healing (Leoni G, neumann PA, sumagin R, denning TL, nusra A (2015) round repair: role of immune-epithelial interactions.8: 959-968.).

Therefore, the gene expression inhibitory effect of the combined extracts of the present invention was determined by real-time quantitative polymerase chain reaction and western blot analysis.

6-1 ribonucleic acid extraction and real-time quantitative polymerase chain reaction

Wound skin tissue of the mice was harvested at day 7 using surgical scissors (PF-24.10, professional, pakistan) 3mm from both sides of the wound. Total ribonucleic acid was extracted by freezing skin tissue in liquid nitrogen (Dongas, inc., korea). Tri-RNA reagent (FATR 001, favorgen, taiwan, china) was added to the frozen skin tissue, and disrupted using beads (D1031-05, bedbug, USA).

0.2mL of chloroform (67-66-3, JUNSEI, japan (Japan)) was added thereto and mixed well, followed by centrifugation at 12000rpm and 4 ℃ for 10 minutes using a centrifuge (5415R, eppendorf, germany).

Only the supernatant was transferred to a fresh microcentrifuge tube (S044378, SARSTEDT AG5CO. KG., germany), 0.4mL of isopropyl alcohol was added thereto and mixed, and the resulting mixture was centrifuged at 12000rpm and 4 ℃ for 20 minutes using a centrifuge (5415R, eppendorf, germany) to precipitate a ribonucleic acid product.

After washing the RNA precipitate with 75% ethanol, the precipitate was centrifuged at 12000rpm at 4 ℃ for 10 minutes. Ribonucleic acid was dissolved in water containing no nuclease (S002, enzynomics, inc., korea).

To this was added recombinant deoxyribonuclease I (DNase I) (M0595, enzynomics, korea), and the mixture was placed in a 37 ℃ incubator (BF-150N, biofree, korea) for 30 minutes. 8M lithium chloride (L9650, sigma Co., USA) was added thereto, and the mixture was left alone overnight at a temperature of-20 ℃.

The next day, after centrifugation at 12000rpm and 4 ℃ for 20 minutes, the ribonucleic acid precipitate was washed with 75% ethanol and centrifuged at 12000rpm and 4 ℃ for 10 minutes. Ribonucleic acid was dissolved in nuclease-free water (S002, enzynomics, inc.) and then quantified.

Complementary deoxyribonucleic acid was synthesized using PrimeScript RT Master Mix (RR 036A, takara, japan) using total ribonucleic acid as a template, and real-time quantitative polymerase chain reaction (qRT-PCR) was performed using the synthesized complementary deoxyribonucleic acid using SYBRgreen kit (RT 500M, enzynomics, korea) and Stratagene Mx3000p (MX 3000p, agilent).

In order to extract a cycle threshold value from cycle threshold values (Cts) of desired genes to obtain a Ct value of 18S and a Ct value of calibration (1/2) ^ the result analysis is performed by the following equation 2. The sequence listing of the primers used is shown in table 10.

Mathematical formula 2

Relative quantitation = (1/2) ^ (desired gene Ct-18S Ct)

Watch 10

Quantification of primer sequences for use in polymerase chain reaction

6-2 Western blot analysis

Wound skin tissue of the mice was harvested at day 7 using surgical scissors (PF-24.10, professional, pakistan) 3mm from both sides of the wound. After the skin tissue was put into phosphate buffer solution, it was washed overnight in a shaker at 4 ℃. The skin tissue was cultured in RIPA buffer (prepared by oneself, 0.1% Sodium Dodecyl Sulfate (SDS), 0.5% sodium deoxycholate, 1% Triton X-100, 2mM ethylenediaminetetraacetic acid (EDTA), 50mM Tris-HCl (pH 8.0), 150mM NaCl) for 30 minutes in ice.

The skin tissue was cut into small pieces with scissors (PF-24.10, professional, pakistan) and disrupted with a microtube homogenizer (985370, DREMEL, mexico). The disrupted skin tissue was subjected to interest separation using a centrifuge (5415R, eppendorf Co., germany) at 13000rpm at 4 ℃ for 10 minutes, and the supernatant was removed.

To this was added 5 Xsample buffer (self-prepared, 1M Tris-HCl (pH 6.8), 50% glycerol, 10% sodium dodecyl sulfate, 2-mercaptoethanol, and 1% bromophenol blue), the tissue was boiled at 100 ℃ for 7 minutes, and cooled in ice for 3 minutes to break off the protein from the sodium dodecyl sulfate polyacrylamide gel. Primary antibodies to matrix metalloproteinase-9 (Millipore, USA, AB 19016), vascular endothelial growth factor-A (abcam, UK, AB 46154), platelet-derived growth factor-A (Santa cruz, USA, sc-9974), beta-tubulin (Santa cruz, USA, sc-166729) were used in the experiments.

As is clear from table 11, the combined extract of the present invention has the effect of reducing the expression of matrix metalloproteinase and the effect of increasing growth factor at the ribonucleic acid and protein levels, and it was confirmed that the test sample group treated with the extract of the present invention has the effect of promoting growth factors (platelet-derived growth factor-a and vascular endothelial growth factor-a) as well as rapidly suppressing the expression levels of various cytokines associated with skin wounds (matrix metalloproteinase-9) and skin ulcers in STZ-induced diabetic mice, as compared to the negative control group treated with distilled water (DIW).

Therefore, it was confirmed that the combined extract of the present invention prepared in the examples had a potent inhibitory effect on skin ulcers and skin wounds and an effect of promoting growth factors that play an important role in the skin proliferation stage.

TABLE 11

Inhibitory Effect on cytokine expression and growth factor-promoting Effect

In particular, platelet-derived growth factors have recently been developed as protein therapeutics for chronic ulcers, vascular endothelial growth factors being vascular growth factors (DiGiovanni CW, petrick JM. The evolution of rh platelet-derived growth factor (PDGF) -BB in mucosalkeletal repirar and its roll in the foot and the foot fusion supply. Foot clone. Clin.2010;15 621-640.DiGiovanni and Petricker, shi R, lian W, han S, cao C, jin Y, yuan Y, zhua H, li M. Nanospere-mediated-delivery of Vascular Endothelial Growth Factors (VEGF) -A and PDGF-B438 for endothelial growth factors in vitamins, genes.8; 201425).

Statistical analysis

The mean and standard error were calculated from the experimental results obtained in the experiment. The significance test was analyzed using the t-test, and the significance (P value) was expressed in terms of P ≦ 0.05=, P ≦ 0.01=, and P ≦ 0.001 =.

Method of the invention

Hereinafter, the formulation and type of the excipient will be described, but the present invention is not limited thereto. Representative preparations are as follows.

Preparing skin care lotion

1.00% of the extract of example (WIN-1001X)

3.00% of Glycerol

1.00% ethanol

0.10% of propylene glycol

Micro-amount spice

Distilled water of less than 100%

The skin preparation is prepared by dissolving the effective components according to the conventional skin lotion preparation method.

Preparation of skin lotion

3.00% of the extract of example (WIN-1002X)

1.00% of magnesium L-ascorbate-2-phosphate

1.00% soluble collagen (1% solution)

0.10% sodium citrate

3.00% of 1, 3-butanediol

Distilled water of less than 100%

The skin lotion is prepared by dissolving the effective components according to a conventional skin lotion preparation method.

Preparation of cream

3.00% of the extract of example (WIN-1003X)

2.00% of polyethylene glycol monostearate

1.00% of glyceryl monostearate

4.00% cetyl alcohol

6.00% squalene

6.00% Tri 2-Glycerol ethylhexanoate

1.00% glycosphingolipid

3.00% of 1, 3-butanediol

Distilled water of less than 100%

A cream is prepared by dissolving the effective ingredients according to a general cream preparation method.

Preparation of facial mask

5.00% of the extract of the example (WIN-1004X)

13.00% polyvinyl alcohol

1.00% of magnesium L-ascorbate-2-phosphate

1.00% of lauroyl hydroxyproline

2.00% soluble collagen (1% solution)

3.00% of 1, 3-butanediol

5.00% ethanol

Distilled water of less than 100%

20g of sugar

20g of fructose

Appropriate amount of lemon flavor

100ml of distilled water

The facial mask is prepared by dissolving the effective ingredients according to a general facial mask preparation method.

Preparing the cosmetic liquid

2.00% of the extract of example (WIN-1005X)

12.00% hydroxyethylcellulose (2% solution)

2.00% Xanthan Gum (2% solution)

3.00% of 1, 3-butanediol

Glycerol at a concentration of 4.00%

5.00% sodium hyaluronate

100ml of distilled water

The cosmetic liquid preparation is prepared by dissolving the active ingredient according to a conventional cosmetic liquid preparation method.

It is therefore evident that the invention as described may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

It should be understood from the above description of the present invention that the same may be modified in various ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Industrial applicability of the invention

As described in the present invention, the present inventors have confirmed that the combined extract of the present invention is very useful for the improvement or treatment of skin ulcer in the form of a topical pharmaceutical preparation or a cosmetic composition, by performing in vitro experiments such as an inhibitory effect on cytokine expression (in vitro) (experimental example 1), a cell growth promoting effect (in vitro) (experimental example 2), and a cell wound healing effect (in vitro) (experimental example 3), and in vivo experiments such as an inhibitory effect on chronic ulcer (in vivo) (experimental example 4), a wound healing effect (in vivo) (experimental example 5), and an inhibitory effect on proinflammatory cytokine expression of growth factors (in vivo) (experimental example 6).

<110> Puyunan

(Zu) Meidi partnering pronunciation

<120> topical composition comprising combination of crude drug extracts containing arillus longan for treating or improving skin ulcer, and use thereof

<130> DIF/2021-02-001/EK

<150> KR 10-2020-0031481

<151> 2020-03-13

<150> KR 10-2021-0022674

<151> 2021-02-19

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Claims (10)

1. A topical pharmaceutical composition characterized by comprising a combined crude drug extract of arillus longan, ligusticum sinense and Polygala tenuifolia Willd as an effective ingredient for treating and ameliorating skin ulcer.

2. A topical pharmaceutical composition according to claim 1,

in the above-mentioned combined crude drug extract,

(a) The mixing ratio of the combined crude drug extracts of arillus longan, rhizoma Ligustici and cortex et radix Polygalae is in the range of 0.01-100: 0.01-100 parts by weight (w/w) based on the dry weight (w/w) of arillus longan, rhizoma Ligustici and cortex et radix Polygalae; or alternatively

(b) The mixing ratio of the extracts of arillus longan, rhizoma Ligustici, and cortex et radix Polygalae is in the range of 0.01-100: 0.01-100 parts by weight (w/w) based on the dry weight (w/w) of arillus longan, rhizoma Ligustici, and cortex et radix Polygalae.

3. The topical pharmaceutical composition of claim 1, wherein the extract is selected from the group consisting of water, C, including methanol, ethanol, propanol, butanol ₁ -C ₄ Extracting with solvent selected from lower alkanol, acetone, ethyl acetate, chloroform, hexane, butanediol, propylene glycol or glycerol.

4. The topical pharmaceutical composition of claim 1, wherein the skin ulcer is selected from the group consisting of decubitus ulcers and diabetic ulcers.

5. A method for treating or ameliorating skin ulcer in a mammal, comprising the step of topically administering to said mammal an effective amount of a combination crude drug extract of arillus longan, ligusticum sinense, and Polygala tenuifolia, and a pharmaceutically acceptable carrier therefor.

6. A use of a combined crude drug extract of arillus longan, ligusticum sinense and Polygala tenuifolia Willd, characterized in that it is used for preparing a topical preparation containing the combined crude drug extract of arillus longan, ligusticum sinense and Polygala tenuifolia Willd as an active ingredient for treating or improving skin ulcer of mammals including human beings.

7. A cosmetic composition characterized by comprising a combined crude drug extract of arillus longan, ligusticum sinense, and polygala tenuifolia as an active ingredient in an amount effective for treating or improving skin ulcer.

8. The cosmetic composition according to claim 7, wherein said extract is selected from the group consisting of water, C including methanol, ethanol, propanol, butanol ₁ -C ₄ Extracting with solvent selected from lower alkanol, acetone, ethyl acetate, chloroform, hexane, butanediol, propylene glycol or glycerol.

9. The cosmetic composition according to claim 7, wherein said skin ulcer is selected from the group consisting of decubitus ulcers and diabetic ulcers.

10. The cosmetic composition according to claim 7, wherein the composition is in a form selected from the group consisting of a skin lotion, a skin softening lotion, a skin toner, an astringent, a skin lotion, an emulsion, a moisturizing lotion, a nourishing lotion, a massage cream, a nourishing cream, a moisturizing cream, a hand cream, a foundation, an essence, a nourishing essence, a mask, a cleansing foam, a cleansing lotion, a makeup removing cream, a skin lotion, a body wash, a hair lotion, and a beauty lotion.

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